Synthesis of single-stranded labelled oligonucleotides of preselected sequence

ABSTRACT

Substantially pure single-stranded oligonucleotides having a preselected sequence of not more than about 200 nucleotides, at least one of which is at a preselected position in the sequence and includes a base with a covalently attached linker arm containing or capable of binding at least one reporter group or solid support. A process for the chemical synthesis of the substantially pure single-stranded oligonucleotide and modified nucleosides useful in such synthesis are provided.

This application is a continuation of application Ser. No. 08/288,337,filed Aug. 10, 1994, now abandoned, which is a divisional of applicationSer. No. 07/505,032, filed Apr. 27, 1990, now abandoned, which is acontinuation of application Ser. No. 07/046,133, filed May 4, 1987, nowU.S. Pat. No. 4,948,882, which is a continuation-in-part of Ser. No.06/617,094, filed Feb. 22, 1984, now abandoned, which is acontinuation-in-part of Ser. No. 06/468,498 filed Feb. 22, 1983, nowabandoned.

BACKGROUND OF THE INVENTION

This invention relates generally to modified oligonucleotides ofpreselected sequence, and more specifically to single-strandedoligonucleotides including nucleotides modified for the attachment ofdetectable reporter groups or solid support.

Nucleic acids, which are the carriers of genetic information betweengenerations, are composed of linearly arranged individual units callednucleotides. Each nucleotide has a sugar phosphate group to which isattached one of the pyrimidine or purine bases, adenine (A), thymine(T), uracil (U), guanine (G) or cytosine (C). In the native state,single-stranded nucleic acids form a double helix through highlyspecific bonding between bases on the two strands; A will bond only withT or U, G will bond only with C. Thus a double stranded nucleic acidwill form where, and only where, the sequence of bases in the twostrands is complementary.

The understanding of complementary bonding between nucleic acids permitsa variety of applications. For example, labelled nucleic acids of knownbase sequence, termed genetic probes, may be used to detect the presenceof complementary nucleic acid in a sample. Such technology provides themost sensitive method available for determining the existence of aparticular gene in a cell or of organisms such as viruses and bacteria.The ideal genetic probe would be of uniform length to allow predictablehybridization behavior and would be of homogeneous sequence to minimizecross reactivity with non-targeted nucleic acids. Moreover, it would besingle-stranded and easily detectable.

One factor which has limited the utilization of genetic probes has beenthe difficulties encountered in producing detectable single-strandednucleic acids having a preselected sequence. Two techniques of nucleicacid synthesis are currently used: enzymatic and chemical, i.e.non-enzymatic. The enzymatic synthesis of nucleic acid requirespreexisting DNA for a template and utilizes natural cellular enzymaticmechanisms to facilitate replication of DNA segments. Two conventionalexamples of such synthesis are the nick translation protocol (Rigby etal., 1977 J. Mol. Biol. 113:237-251) and the gap-filling reaction(Bourguignon et al., 1976. J. Virol. 20:290-306). In both methods,preexisting DNA is contacted with an enzyme known as a DNase which nicksthe strand, causing a break between a 3'-hydroxyl group and the adjacent5'-phosphate. Such nicked or gapped DNA then serves as both a templateand a primer. In the presence of a DNA polymerase, such as POL I whichis isolated from E. coli, free nucleotides are successively condensed onto the 3' hydroxyl group while nucleotides adjacent to the 5' end of thenick are simultaneously cleaved. Double-stranded DNA having new strandscomposed of the added nucleotides is thus formed. Although DNApolymerases are the enzymes most commonly used in such procedures, otherenzymes such as terminal transferases, reverse transcriptases and RNApolymerases can also be used with similar results.

If one or more of the provided nucleotides are modified, for example toinclude a label, such modifications will be incorporated into the newstrand. Only a limited array of modifications may be utilized in such amethod, however, due to the interference of the modifications with theactivity of the enzymes. Radioisotopes, such as ³² P or ¹⁴ C, may bereadily incorporated since they closely resemble the natural isotopes,and thus radioactively labelled probes have been widely used. Because ofthe potential hazards associated with handling and disposing ofradioactive materials and their inherent instability, however,radioactive probes are undesireable.

Certain other modified bases have been incorporated intooligonucleotides prepared by enzymatic synthesis. Ward et al., EuropeanPatent Application No. 82301804.9 disclose pyrimidine and purine baseshaving certain moieties attached, such as biotin, which are capable ofcomplexing with a polypeptide for detection. These modified bases can beincorporated into enzymatically produced nucleic acids. However,hybridization probes produced by such methods have inherent drawbackswhich limit their usefulness. For example, enzymatic synthesis relies onnicked preexisting DNA to serve as a template. Because multiple nicksare introduced randomly in a single chain by contact with a DNase,double-stranded oligonucleotides of widely different composition,sequence, and length will be simultaneously produced. Length of productchains vary considerably, usually from 400 to 1000 bases in length. Ingeneral, chains of over 200 bases are termed "polynucleotides" whilethose under 200 bases are termed "oligonucleotides." Such enzymaticsynthesis chains may reach a length of several thousand nucleotides butfor practical purposes, they generally cannot be made less than abouttwo hundred nucleotides in length. The absolute length cannot becontrolled, however, and the product will be a heterologous mixture oflengths and sequences. No conventional method permits separation andpurification of these heterogeneous pieces. Moreover, it is not possibleto control the site at which the modified nucleotide is incorporatedinto the newly formed chain. While the identity of the particularnucleotide which is modified does determine that the label will beincorporated opposite a position of the complementary nucleotide in thetemplate, the method does not permit the synthesis of a polynucleotidehaving modifications at particular preselected sites among thoseavailable. More importantly, the range of modifications of thenucleotides which can be incorporated is limited to those which will berecognized and incorporated by the enzymes.

To a limited extent, modifications to nucleic acids have also beenintroduced by post-synthetic modification of an enzymaticallysynthesized nucleic acid, such as by mercuration or palladium catalyzedaddition reactions. Only cytosine residues are susceptible to suchaddition reactions, however; thymine and purine bases cannot be modifiedby this method. Moreover, as with enzymatic incorporation of modifiedbases, the particular site at which the modification may be introducedcan not be preselected and both strands are randomly modified. Where thestoichiometry of the reaction is controlled so as to modify only alimited proportion of the available nucleotides, the modifications willbe introduced randomly at sites appropriate to cytosine incorporation,thereby producing a heterogeneous population of modified nucleic acids;see Bigge, et al., (1981). J. Carb. Nucleosides Nucleotide, 8:259(1981). Furthermore, it has been demonstrated that where the reactionconditions are intensified so as to modify substantially all availablenucleotides, undesired chemical degradation of the oligonucleotideensues. Dale et al., (1975). Biochem. 14:2447. This method has not beenused to incorporate labels or reporter groups.

The prior art methods of enzymatic synthesis require double-stranded DNAas a template, and produce double-stranded nucleic acids having labelincorporated in both strands. Moreover, the resulting nucleic acids areheterogenous, varying both in sequence, length, and in position of themodified bases. Enzymatic synthesis cannot produce a single strandedprobe of preselected length, preselected sequence having unique reportergroups defined by site and number. Furthermore, the scope ofmodifications obtainable in the oligonucleotide product is severelyrestricted as the enzymes required for modification can only recognizeand incorporate a very limited array of modified nucleotides in bothstrands of a double-stranded, nonuniform nucleic acid. As a resultproteins, nucleic acids, carbohydrates, fluorophors, and lumiphorscannot be incorporated as labels by these methods.

Naturally occurring nucleotides may be condensed into single-strandedoligonucleotides of preselected sequence and length using chemical, ornon-enzymatic, methods of synthesis. Such methods have been reviewed byMatteucci, et al., (1982). J. Amer. Chem. Soc. 103:3185. Chemicalsynthesis usually involves successive coupling of an activatednucleotide monomer and a free hydroxyl-bearing terminal unit of agrowing nucleotide chain. The coupling is effected through a reactivephosphorous-containing group, such as a phosphate diester or more often,a phosphite triester. Phosphochloridite (Letsinger, et al., (1980). J.Org. Chem. 45:2715) and phosphoamidite (Caruthers, et al., U.S. Pat. No.4,458,066) reactions are commonly used. Caruthers teaches the synthesisof oligonucleotides containing as many as 30 bases composed of onlynaturally-occurring nucleotides. However, no chemical synthesis ofoligonucleotides incorporating modified bases or reporter groups of anytype has been disclosed in the prior art.

Accordingly, there exists a long felt and compelling need forsingle-stranded oligonucleotides of preselected sequence and lengthhaving incorporated therein modified nucleotides capable of detection.Such modifications should be non-radioactive and preferably allowaccurate and inexpensive detection. The present invention satisfies thisneed and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides a substantially pure single-strandedoligonucleotide comprising a preselected sequence of not more than about200 nucleotides, at least one nucleotide of which located at apreselected position in the sequence, including a base with a covalentlyattached linker arm containing at least one reporter group or a solidsupport or a moiety capable of binding at least one reporter group or asolid support. The linker arm can be attached to the base at anyposition, including the C-5 position when the base is a pyrimidine andthe C-8 position when the base is a purine. The reporter group can becapable of colorimetric, fluorescent, luminescent or antibody- or otherligand-mediated detection. The reporter group could also be aradioactive moiety. The oligonucleotide can be either anoligodeoxyribonucleotide or an oligoribonucleotide.

The invention further provides a process for the chemical synthesis ofsuch substantially pure single-stranded oligonucleotides having at leastone modified nucleotide which comprises the stepwise addition ofreactive nucleotides in a preselected sequence to a substantially puresingle-stranded oligonucleotide of less than 200 nucleotides, alsohaving a preselected sequence. The addition is accomplished through areactive phosphorous-containing group attached to a 3'- or a 5'-hydroxylgroup. At least one of the nucleosides so added contains a base having acovalently attached linker arm containing, or capable of binding, areporter group or solid support. The invention also provides modifiednucleoside monomers useful in the above process.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a substantially pure single-strandedoligonucleotide of preselected sequence, at least one nucleotide ofwhich has a substituent group or linker arm which has bound or iscapable of binding a detectable reporter group or solid support. Theoligonucleotide of the present invention is non-enzymaticallysynthesized by the stepwise addition of a selected reactive nucleotidemonomer and a free hydroxyl-bearing terminal unit of an oligonucleotidechain of preselected sequence, at least one nucleotide of the completedchain having a substituent group bound or capable of binding at leastone reporter group or a solid support. The invention also providesreactive nucleotide monomers useful for the synthesis of theabove-described oligonucleotides. Such monomers contain an activephosphorous-containing group at the 5'- or 3'-hydroxyl of aribonucleoside or deoxyribonucleoside, a linker arm attached to the baseand bound to or capable of binding a reporter group or solid support,and appropriate blocking groups on reactive sites. The incorporation ofone or more such modified nucleotide into an oligonucleotides results ina modified oligonucleotide.

The nucleotide units in the modified oligonucleotide of the presentinvention can be purine or pyrimidine based, and can be ribonucleotidesor deoxyribonucleotides. Such bases can take the form of the purinesadenine (A), quanine (G) and hypoxanthine (H), or of the pyrimidinesuracil (U), cytosine (C) or thymine (T). When reference is made hereinto the use of purine or pyrimidine bases, such expressions are intendedto include analogs of such bases. Among such analogs are the analogs ofpurine bases, such as amino-, aza- or deaza-adenosines (tubercidins,formycins, hypoxanthines, and the like), and the analogs of pyrimidinebases, such as deazauracil, deazacytosine, azauracils, azacytosines, andthe like. Alternatively, any other base isolated from natural sourcesmay be used.

The substituent groups or linker arms of the present invention which arecapable of binding reporter groups can be generally characterized asnucleophilic. Exemplary of such linker arms are those which contain atleast one reactive amino, carboxyl, hydroxyl and thio groups and thelike.

In a preferred embodiment, the linker arm is attached to a stericallytolerant site on a nucleotide. A sterically tolerant site is defined asone where the attachment of the substituent group will not causesignificant interference with either the hybridization of the modifiedoligonucleotide to a complementary nucleic acid segment or with thebinding of the linker arm to a reporter group. Such sterically tolerantsites are found, for example, at C-8 position of a purine and the C-5position of a pyrimidine base. Nucleotides having substituent groupsbound at sites other than those which are sterically tolerant are alsouseful, however. For example, where only a portion of the probe isdesigned to hybridize with a targeted DNA segment, the linker arm may belocated external to the area of complementarity without interfering withhybridization. Moreover, even if the linker arm is located within thehybridizing segment so as to prevent binding of the particularnucleotide to which it is attached, hybridization of the surroundingnucleotides may be sufficient to provide a useful probe.

A reporter group can be defined as a chemical group which has a physicalor chemical characteristic which can be readily measured or detected byappropriate detector systems or procedures. Ready detectability can beprovided by such characteristics as color change, luminescence,fluorescence, or radioactivity; or it can be provided by the ability ofthe reporter group to serve as a ligand recognition site. Suchcharacteristics can be measured or detected, for example, by the use ofconventional colorimetric, spectrophotometric, fluorometric orradioactivity sensing intrumentation, or by visual inspection.

The interactions which can be usefully initiated by the reporter groupdefined herein include appropriately specific and selective interactionsproductive of groups or complexes which are themselves readilydetectable, for example, by colorimetric, spectrophotometric,fluorometric, or radioactive detection procedures. Such interactions cantake the form of protein-ligand, enzyme-substrate, antibody-antigen,carbohydrate-lectin, protein-cofactor, protein-effector, nucleicacid-nucleic acid and nucleic acid-ligand interactions. Examples of suchligand-ligand interactions include dinitrophenyl-dinitrophenyl antibody,biotin-avidin, oligonucleotide-complementary oligonucleotide, DNA-DNA,RNA-DNA and NADH-dehydrogenase. Either one of each of such ligand pairsmay serve as a ligand recognition type reporter group.

In the process of the present invention, a selected reporter group orgroups can optionally be attached to the nucleotide monomer beforecoupling of the monomer to the terminal unit of the nucleotide chain, orcan be attached to the product oligonucleotide after formation thereof.The sequence of the nucleotides in the product oligonucleotide ispreselected to provide such oligonucleotide with the specificitynecessary to hybridize with the targeted sequence.

The coupling step preferably involves coupling of a selected monomerunit activated at the 3' position with a free 5'-hydroxyl of theterminal unit of the growing nucleotide chain. Alternatively, suchcoupling can involve attaching a monomer unit activated at the 5'position with a free 3'-hydroxyl of the terminal unit of the nucleotidechain. The terminal unit can be the initial or only unit in the growingnucleotide chain at the time of coupling of the modified nucleotidemonomer, or it can be the terminal nucleotide of an oligonucleotidehaving a preselected sequence.

The term "substantially pure," as used herein, is intended to includethose oligonucleotides which have the preselected sequence ofnucleotides and have linker arms at preselected locations in thesequence. Oligonucleotides of divergent sequence are excluded. However,the term is specifically intended to include those oligonucleotidescomprising subsequences of the predetermined sequence which result fromincomplete coupling of reactive nucleotides into the growingoligonucleotide chain. In practice, even under ideal conditions lessthan all molecules present will exist as the desired product. Forexample, when coupling a nucleotide monomer and a hexamer into aseptamer, three moieties will be present after the reaction mixture iscomplete; the heptamer, the hexamer and the nucleotide monomer. Whilethe majority of the reaction mixture will exist as the desired heptamer,a certain proportion will inevitably remain as one of the startingreagents. If necessary, these reagents can be separated from the desiredoligonucleotide product by well-known methods such as high performanceliquid chromotography and gel electrophoresis. Nonetheless, the presenceof these artifacts of incomplete reaction are not intended to defeat thesubstantially pure nature of the modified oligonucleotide of the presentinvention. Furthermore, it is understood that the presence of unrelatedchemical moieties such as solvents, buffers and other non-nucleic acidcomponents do not diminish the substantially pure nature of theoligonucleotides.

The substantially pure single-stranded oligonucleotides of the presentinvention are useful tools in protocols involving nucleic acidhybridization techniques. Among such uses are the identification,localization, isolation or quantification of complementary nucleotidesequences of interest in cellular or cell-free systems. Such protocolscan be used, for example, to detect the presence of nucleotide sequenceswhich are either native or the result of infective agents. Infectiveagents can include viruses, bacteria, mycoplasmas, parasites, includingChlamydia and Rickettsia, and fungi. Such uses can also includediagnostic applications of any fundamental biological event detectablethrough hybridization of nucleic acid components, or immobilization orpurification of complementary sequences by affinity chromatography whenthe product oligonucleotide is attached to a solid support.

The non-enzymatic synthetic process of the present invention producespreferred oligonucleotides of the following generic formula: ##STR1##wherein n is 1 to about 199, preferably about 5 to about 60, and mostpreferably about 10 to about 40, R' is hydrogen or hydroxy, and B is anyone of the purine or pyrimidine bases adenine, guanine, hypoxanthine,cytosine, uracil, thymine, or any other naturally occurring base, thenucleotide units having naturally occurring bases being independentlyintermixed with one or more nucleotide units having modified bases(B^(m)). The modified pyrimidine bases (Py^(m)) are substitutedpreferably but not exclusively at the C-5 position, and typical examplesthereof are the uracil and cytosine bases illustrated by the followinggeneric formulas: ##STR2## The modified purine bases (Pu^(m)) arepreferably but not exclusively substituted at the C-8 position, andtypical examples thereof are the modified adenine and guanine basesillustrated by the following generic formulas: ##STR3##

The substituent group or linker arm R is characterized by its ability tobind or be composed of one or more reporter groups or solid supports. Inthe modified pyrimidine bases the linker arm R generally comprises twoor more carbon atoms, whereas in the modified purine bases R generallycomprises one or more carbon atoms. In this context, R preferably takesthe form of one of the following functionalized carbon chains: ##STR4##wherein R₁ is hydrogen, or alkyl; R₂ is alkyl, alkenyl, aryl, orfunctionalized alkyl, alkenyl, aryl wherein functional groups includeone or more amines, amides, nitriles, carboxylic acids and esters,hydroxyls, sulfonates, or the like; and Z is a polyvalent heteroatomsuch as nitrogen, oxygen or sulfur.

R₂ contains the site of the attachment of the bases to a solid support,or to one or more reporter groups which function, for example, as acolorimetric, fluorescent, luminescent, radioactive, or ligandrecognition group. Functionally fluorescent groups include fluoresceins,rhodamines, and the like, or proteins capable of producing fluorescence;functionally luminescent groups include luminols, acridines, luciferins,dioxetanes, dioxamides, and the like, or proteins capable of producingluminescence. Ligand recognition groups include vitamins (such as biotinor adducts thereof, including iminobiotin and desthiobiotin), antigenssuch as dinitrophenyl, amino benzenesulfonates, carbohydrates and otherfunctional groups or adducts of such groups which can be recognized byligand-like interactions with proteins, or from which such ligand-likeinteractions can be elicited. A second oligonucleotide capable ofspecific interaction with the first oligonucleotide is illustrative of agroup from which a ligand-like interaction can be elicited. Ligandrecognition groups can also serve as functionally colorimetric reportergroups when recognition results in dye formation. For example, whendinitrophenyl is used as a reporter group, known detection systems usingan anti-dinitrophenyl antibody coupled to peroxidase can be used as adetection system, resulting in a color change. Functionally radioactivegroups incorporate a radioactive element in the chosen reporter group,or a protein capable of producing a useful radioactive product.

Oligonucleotides of Formula I are prepared by chemical synthesis frommonomer nucleotide analog units of the formula: ##STR5## wherein R₃ istrityl(triphenylmethyl), dimethoxytrityl, or another appropriate maskinggroup for the 5'-hydroxyl; B and R' are masked, if appropriate; and Prepresents a phosphorus-containing group suitable for internucleotidebond formation during chain extension in synthesis of a productoligonucleotide. The phosphorus-containing groups P suitable forinternucleotide bond formation are preferably alkylphosphomonochloridites or alkyl phosphomonoamidites. Alternativelyphosphate triesters can be employed for this purpose. The monomer unitcan alternatively have R₃ attached at the 3'-hydroxyl and P attached atthe 5'-hydroxyl.

Generally, the term "masking group" or "blocking group" is a functionalexpression referring to the chemical modification or "blocking" of anintegral functional group by attachment of a second moiety to disguisethe chemical reactivity of the functional group and prevent it fromreacting in an undesired manner during reactions at other sites in themolecule. Such modification is reversible, and allows subsequentconversion back to the original functional group by suitable treatment.In many cases, such masking formally interconverts structuralfunctionality, e.g., a primary amine masked by acetylation becomes asubstituted amide which can be later converted back to the primary amineby appropriate hydrolosis.

The compounds of Formula I include the acceptable conjugate acid saltsthereof. Conjugate acids which may be used to prepare such salts arethose containing nonreactive cations and include, for example,nitrogen-containing bases such as ammonium salts, mono-, di-, tri-, ortetra-substituted amine salts, and the like, or suitable metal saltssuch as those of sodium, potassium, and the like.

The process steps of the present invention will now be generallydescribed and illustrated diagrammatically. Thereafter, the inventionwill be illustrated more specifically and detailed examples thereofprovided. Since the invention relates to oligonucleotides incorporatingboth pyrimidine-based and purine-based nucleotide units, the use of bothpyrimidine and purine-based compounds in the synthetic process will beillustrated. The specific pyrimidine and purine-based compoundsillustrated are only exemplary of the respective pyrimidine and purineclasses, and it is to be understood that any other member of therespective class can be substituted therefore in the process and theproduct oligonucleotide, whenever suitable or desired. Whiledeoxyribonucleotide compounds are shown for the most part, it isunderstood that ribonucleotide compounds are also contemplated by theinvention and can be substituted for the deoxyribonucleotide compoundswherever ribonucleotide compounds are desired in the productoligonucleotide.

The reactive nucleotide monomers of this invention are essential asintermediates in the process for synthesizing the new oligonucleotides.These reactive nucleotide monomers are represented by the followingstructure ##STR6## Wherein B is a pyrimidine or purine base; R is alinker arm containing or, when unblocked, capable of binding at leastone reporter group or a solid support; and provided that when R₄ is amasking group, then R₅ is either a reactive phosphorous-containing groupor H if the 5'-OH group of the 5'-terminal nucleotide of a growingoligonucleotide contains a reactive phosphorous-containing group; andwhen R₅ is masking group then R₄ is either reactivephosphorous-containing group or H if the 3'-OH of the 3'-terminalnucleotide of a growing oligonucleotide contains a reactivephosphorous-containing group; and R₈ is H or a masked hydroxyl group.

Such reactive nucleotide monomers each have a base which is modified bya linker arm comprising a functionalized carbon chain incorporating atleast the functional group as aforedescribed, preferably including oneor more amides, the nitrogen of the amides being preferably attached toa sterically tolerant site on the base through the carbon chain. In thecase of pyrimidine-based nucleotides, the carbon chain is preferablyattached at the C-5 position, and in the case of the purine-basednucleotides, the carbon chain is preferably attached at the C-8 positionthough a polyvalent heteroatom, such as nitrogen, oxygen or sulfur. Inaddition such nucleotides are chemically blocked at the 5' position (orthe 3' position) with a group, such as dimethoxytrityl, appropriate forthe chemical synthesis of oligonucleotides.

In the new class of nucleotide the linker arm R can be chosen from##STR7## wherein R₁ is hydrogen or C₁₋₆ lower alkyl, n is 0 to 20 and Ycontains at least one blocked amino or blocked carboxyl or blockedhydroxy or blocked thio group or at least one reporter group or solidsupport (i.e. C_(n) H_(2n) Y=R₂). More specifically, Y can include oneor more dinitrophenyl, or ##STR8## wherein X is hydrogen, fluorine orchlorine. The amino, carboxyl, hydroxy, and thio groups in Y are blockedbecause of the presence of the active phosphorous-containing groups atpositions R₄ or R₅. Synthesis of these nucleosides, as well as of themasked forms thereof, is described hereinafter in Examples I, throughXV, XXIX, XXX.

Preferred nucleosides incorporate the substituent group ##STR9## at C-5of pyrimidine nucleosides wherein n=3 to 12 and Y is ##STR10## Mostpreferred are such nucleosides wherein the pyrimidine base is uracil.

The process of the present invention for preparing the modifiedoligonucleotide can be initiated by the preparation of the selectednucleoside. Generally, the most preferred nucleosides are best preparedin the following manner. 5-(Methyl 3-acrylyl)-2'-deoxyuridine isprepared from 2'-deoxyuridine by the method of Bergstrom and Ruth,(1976). J. Amer. Chem. Soc. 96:1587. The nucleoside is then treated with1.05 equivalents of dimethoxytrityl chloride in pyridine for 4 hours toblock the 5'-hydroxyl with dimethoxytrityl (DMT). The resulting productis purified by silica chromatography eluting a gradient of 0-10%methanol in chloroform containing 2% triethylamine. The purified5'-DMT-5-(methyl 3 acrylyl)-2'-deoxyuridine is treated with 1N KOH for24 hours at ambient temperature to hydolyze the methyl ester. Theresulting 5'-DMT-5-(3-acrylyl)-2'-deoxyuridine is treated with excessdicyclohexylcarbodiimide and hydroxybenztriazole in pyridine. After 4hours, a 2 to 5 fold excess of 1,7-diaminoheptane is added, and thereaction stirred overnight. After 12 to 20 hours, a 10 to 20 fold excessof trifluoroacetic anhydride is added, and the reaction stirred at roomtemperature for 4 hours. The product is purified by silicachromatography eluting a gradient of 0 to 10% methanol in chloroformcontaining 2% triethylamine, followed by exclusion chromatography usingSephadex LH-20 eluting 1% triethylamine in methanol. Appropriatefractions are combined to yield5'DMT-5-[N-(7-trifluoroacetylaminoheptyl)-1-acrylamido]-2'-deoxyuridine;such product is appropriate for oligonucleotide synthesis by thephosphochloridite procedure described in Examples XV and XVIII.Alternatively, such a compound can be prepared by the combination ofmethods described in Examples II and III. Replacing diaminoheptane inthis process with other diamino-alkanes (e.g., diaminopropane,diaminohexane, diaminododecane) is productive of other compounds ofvarying substituent length wherein n=3, 6, or 12 and R= ##STR11## Twosuch nucleosides, one pyrimidine (uracil)-based and the other purine(adenine)-based, are shown at the top of the diagram below illustratingthe process. Reactive sites on the bases of the nucleosides are thenmasked, as shown in Reaction 1, by attachment of, for example, a benzoylgroup (Bz) to the amine at the 6 position of the adenine-basednucleoside. Such masking is generally described in "Synthetic Proceduresin Nucleic Acid Chemistry", Vol. 1, W. Zorbach and R. Tipson eds.(Wiley-Interscience, New York) (1968). Unprotected amines on thesubstituent group are masked, for example, by attachment thereto oftrifluoroacetyl groups (Ac), as also shown in Reaction 1.

It is important for the purposes of this invention to have appropriateblocking or masking groups for the functional moiety on the linker arm.These blocking groups must be substantially stable towards all chemicalsteps used in the synthesis of the oligonucleotide, but be capable ofselective removal or deblocking, without degradation of the functionalmoiety or the oligonucleotide.

The selected 3'- or 5'-hydroxyl of the nucleoside is then masked byattachment thereto of a dimethoxytrityl (DMT) group. In Reaction 2illustrated below, the 5'-hydroxyl is masked, leaving the 3'-hydroxylfree or available for reaction. Alternatively, the 3'-hydroxyl could bemasked, leaving the 5'-hydroxyl free.

The nucleoside is then converted to an activated nucleotide monomer,preferably by attachment to its 3' hydroxyl of a phosphorus-containinggroup which includes an activating moiety. When the modified nucleosideis properly blocked, modifications of the procedures described byLetsinger, et al., Matteucci, et al., or as reviewed by Narang, et al.can be utilized for oligonucleotide synthesis. The use ofphosphochloridite chemistry such as that disclosed by Letsinger, et al.,is detailed in Examples XVI-XVIII. In order to use phosphoamiditechemistry, a modification of the procedure of Beaucage and Caruthers isused as described in Examples XXIX and XXX, by phosphitylating theprotected modified nucleoside with methyl chloro(N,N-diisopropyl)phosphoamidite or methyl chloro phosphomorpholidite, asin the improved procedure of Dorper, et al. (1983). Nucleic Acids Res.11:2575. Alternatively, the protected modified nucleoside can bephosphorylated with 1.2 eq. chlorophenyl dichlorophosphate intrimethylphosphate at room temperature followed by quenching with waterto give the 3'-chlorophenyl phosphate adduct of the modified nucleoside,such adducts being useful in a modification of the phosphotriesterapproach as illustratively reviewed by Narang, et al. The diagramillustrates in Reaction 3 the synthesis of activated monomer nucleotideunits of Formula II by attachment to the nucleoside 3'-hydroxyl of aphosphomonochloridite group in which the chlorine functions as anactivating moiety.

Coupling or condensation of the selected activated nucleotide monomer,i.e. the uracil-based monomer or the adenine-based monomer, to theterminal unit of a growing nucleotide chain is illustrated in Reaction 4in the diagram. The nucleotide chain is shown as including in its righthand end a nucleotide unit having a naturally occurring base and havinga solid support or masking group R₄ attached to its 3'-hydroxyl. Theillustrated chain also includes one or more (n') nucleotide units havingnaturally-occurring bases, said units being coupled to the 5'-hydroxylof the nucleotide unit, the terminal of one of the nucleotide unitshaving a free hydroxyl at the 5' position. In the coupling reaction thechlorine of the monomer reacts with the hydrogen of the free hydroxyl ofthe terminal unit and is displaced, so that the oxygen of the terminalunit couples to the phosphorus of the monomer as shown, and the monomerthereby becomes the new terminal unit of the nucleotide chain.

The DMT 5' blocking group is then removed to permit further extension ofthe nucleotide chain by sequential coupling thereto of additionalactivated nucleotide monomer units. The nucleotide units added to thechain can be preselected and may have either naturally occurring ormodified bases. The diagram shows in Reaction 4a the further extensionof the chain by the addition of one or more (n") nucleotide units havingnaturally occurring bases.

When an oligonucleotide of the selected length and sequence has beensynthesized, the DMT group can be removed from the terminal unitthereof, and the masked reactive groups are unmasked. Examples ofmodified uracil and adenine bases with their reactive groups unmaskedare also shown diagrammatically at Reaction 5. If the initial nucleotideunit of the chain is bound to a solid support R₄, the chain is thengenerally removed from such solid support. The appropriate order ofunmasking can be preselected.

Reporter groups R₅ appropriate for the intended use of the productoligonucleotide can then be bound to such substituent groups asexemplified in Reaction 6, which illustrates the respective bases withreporter groups R₅ bound to the respective substituent groups thereof.##STR12##

Having discussed the process of the present invention in general termsand illustrated the same diagrammatically, each of the reactionsreferred to will now be discussed more specifically.

With reference to Reaction 1, masking of chemically reactive amines suchas N⁴ of cytosine, N⁶ of adenine, N² of guanine, and alkyl or arylamines of the modified bases with suitable masking groups can beconveniently accomplished in suitable solvents such as alcohols,pyridines, lutidines, chloroform, and the like, by reaction of thenucleosides with an excess of appropriate acid anhydrides for about 1 to24 hours at temperatures in the range of 0° C. to 110° C., generally 20°C. to 80° C. Appropriate acid anhydrides include acetic anhydridetrifluoroacetic anhydride, benzoyl anhydride, anisoyl anhydride, and thelike. Preferred are acetyl, trifluoroacetyl, benzoyl, and isobutyrylanhydride.

Masking of the 5'-hydroxy in Reaction 2 can be conveniently effected byreaction of the nucleosides with a slight excess of appropriateacid-labile masking reagents, such as tritylchlorides, monomethoxytritylchloride, dimethoxytrityl chloride (DMTC1), trimethoxytrityl chlorideand the like. Preferred is dimethoxytrityl chloride. Typical reactionsare carried out in suitable solvents, such as pyridine, lutidines,trialkylamines, and the like at temperatures in the range of -20° C. to120° C., generally 20° C. to 100° C., for about 1 to 48 hours. Thepreferred reaction utilizes 1.1 equivalents of DMTC1 in pyridine at roomtemperature for 2 hours.

It is generally preferred that the respective products of each reactiondescribed hereinabove be separated and/or isolated prior to use as astarting material for a subsequent reaction. Separation and isolationcan be effected by any suitable purification procedure such as, forexample, evaporation, filtration, crystallization, columnchromatography, thin layer chromatography, etc. Specific illustrationsof typical separation and isolation procedures can be had by referenceto the appropriate examples described hereinbelow; however, otherequivalent separation procedures can, of course, also be used. Also, itshould be appreciated that, where typical reaction conditions (e.g.,temperatures, mole ratios, reaction times) have been given, conditionsboth above and below the typical ranges can also be used, thoughgenerally less conveniently.

Activation to the phosphite analog illustrated in Reaction 3 can be mostconveniently effected by treatment of the nucleoside compounds withsuitable phosphitylating agents in appropriate solvents at temperaturesin the range of -90° C. to 60° C. for 1 minute to 2 hours. Suitablephosphitylating agents include methylphosphodichloridite,o-chlorophenylphosphodichloridite,p-chlorophenylphosphosphodichloridite,methylphospho(dialkylamino)monochloridite, and the like. Appropriatesolvents include pyridine, lutidines, acetonitrile, tetrahydrofuran,dioxane, chloroform and the like containing 0-20% appropriate base(generally 1-5 vol %) such as lutidines, collidines, triakylamines andthe like. Preferred phosphitylating agents aremethylphosphodichloridite, o-chlorophenylphosdichloridite, andmethylphospho (di-iso-propylamino)-monochloridite. One example of suchphosphytilating conditions are with 0.9 equivalents ofmethylphosphodichloridite in pyridine or acetonitrile containing 5%2,6-lutidine for 5 to 10 minutes at room temperature or below.

The chemical incorporation of the modified nucleotide analog monomersinto a growing nucleotide chain to produce a single strandoligonucleotide is illustrated in Reactions 4 and 4a. Typicalcondensations are in appropriate solvents at temperatures in the rangeof -20° C. to 50° C., preferably at ambient temperature, for about 0.5to 60 minutes. Appropriate solvent mixtures include pyridine, lutidines,acetonitrile, tetrahydrofuran, dioxane, chloroform and the likecontaining 0-20% appropriate base (generally 1 to 5 volume %) such aslutidines, collidines, trialkylamines and the like) for the chloriditemethod, or with a suitable activator such as 0-20% tetrazole for theamidite methods. The growing chain may be soluble, insoluble, orattached to a suitable solid support by appropriate chemical methodsknown in the art. Preferred is attachment to a solid support.Furthermore, the growing chain may or may not have previouslyincorporated one or more modified nucleotide analogs.

After condensation of the activated monomer to the growing chain, inReaction 4, the initial product can be treated with suitable reagents toaccomplish oxidation of the intermediate phosphite triester, optionalcapping to block unreacted 5'-hydroxyls on the oligonucleotide chain,and removal of the 5'-DMT group. Oxidation of the phosphite triester canbe accomplished by treatment with 0.1-5 w/vol % iodine in suitablesolvents, for example, tetrahydrofuran/water/lutidine mixtures. Chemicalcapping of unreacted 5'-hydroxyls can be accomplished by acetylation oracylation with, for example, acetic anhydride and4-dimethylaminopyridine in tetrahydrofuran/lutidine mixtures. Removal ofthe 5'-blocking group, usually DMT, is most conveniently effected bytreatment with mild organic acids in nonprotic solvents, such as mildacids including, for example, 1-5 vol % dichloroacetic ortrichloroacetic acid in chloroform or dichloromethane. The growingnucleotide chain, after removal of DMT, can now serve as acceptor forsubsequent elongation by sequential reaction with activated monomers toeventually produce the oligonucleotide of desired length and sequence,as shown in Reaction 4a.

After an oligonucleotide of desired sequence is produced, Reaction 5 isaccomplished to provide the product oligonucleotide. To this end,thiophenol treatment is used to remove methyl masking groups fromphosphate triesters, and suitable aqueous alkali or ammonia treatment isused to remove other masking groups from the phosphate triester andbenzoyl, acetyl, isobutyl, trifluoroacetyl, or other groups from theprotected amines and/or to remove the product from the solid support.Removal of DMT from the oligonucleotide product is accomplished by theappropriate treatment with a mild acid, such as aqueous acetic acid atambient temperature to 40° C. for 10 to 60 minutes. Such reactions maybe accomplished before or during final purifications. Final purificationis accomplished by appropriate methods, such as polyacrylamide gelelectrophoresis, high pressure liquid chromatography (HPLC), reversephase or anion exchange on DEAE cellulose, or combinations of thesemethods.

The process described herein for synthesis of oligonucleotides canutilize modified deoxyribonucleosides (where R' is H) or modifiedribonucleosides (where R' is hydroxyl). When ribonucleosides are used,the 2'-hydroxyl is masked by an appropriate masking group such as, forexample, that afforded by silylethers or tetrahydropyran. Other riboseanalogs, including arabinose and 3'-deoxyribose, can also beaccommodated in the process to produce the desired oligonucleotide.

The linker arm modifying a nucleotide base must be capable of bindingone or more reporter groups or solid supports either prior to or afterthe chain extension coupling reaction. In the latter case, selectedproduct oligonucleotides are reacted with suitable reagents to attachsuch reporter groups. For example, when modified bases are incorporatedinto the oligonucleotide and R₂ of the linker arm contains one or moreprimary amines, coupling with amine-reactive groups such as isocyanate,isothiocyanate, active carboxylic acid derivatives, epoxides or activearomatic compounds using suitable mild conditions is productive ofamide, urea, thiourea, amine or aromatic amine linkages. For example, anoligonucleotide which contains an uracil or adenine base modified by alinker arm having a primary amine, as shown in the Reaction 5 diagram,can be reacted with a suitable reagent, such as fluoresceiniso-thiocyanate (FITC) or N-hydroxysuccidimidyl 6-biotinylaminocaproicacid to provide a reporter group R₅ (fluorescein or biotin,respectively) bound to the linker arm as shown in Reaction 6. Otherreporter groups which can be attached in similar manner include a widevariety of organic moieties such as fluoresceins, rhodamines, acridiniumsalts, dinitrophenyls, benzenesulfonyls, luminols, luciferins,carbohydrates and the like, or proteins capable of producing detectableproducts. Suitably active reporter groups are available commercially, orcan be synthesized, for example, by processes of the type generallydescribed in "Bioluminescence and Chemiluminescence" [M. Denuca and W.McElroy, eds., Acad. Press, New York (1981)], by D. Roswell, et al., orH. Schroeder, et al. [Meth. Enzymol. LXII, 1978], and references citedtherein.

Typically, attachment of reporter groups is conveniently accomplished inpredominantly aqueous solvents by reaction of the substituent groups ofmodified bases wherein R₂ =C_(n) H_(2n) NH₂ with excess of the selectedreporter group at temperatures in the range of about -20° C. to 50° C.(preferably 20° C. to 40° C.) for 1 to 24 hours. Suitable solvents arean aqueous buffer and 0-50% organic solvents such as lower alcohols,tetrahydrofuran, dimethylformamide, pyridine, and the like. Preferredreporter group reactants include fluorescein, isothiocyanates,dinitrophenylisothiocyanates, fluorodinitrobenzene,N-hydroxysuccinimidylbiotin, N-hydroxysuccinimidyl dinitrobenzoate,isothiocyanates such as aminobutyl ethyl isoluminol isothiocyanate andthe like, active esters of carboxyfluorescein, rhodamine, biotinadducts, dioxetanes, dioxamides, carboxyacridines, carbohydrates and thelike, and suitably activated proteins.

Additionally, when the product oligonucleotide includes modified baseswherein R contains one or more carboxylic acids, mild condensationswith, for example, primary alkylamines is productive of amide linkages.Typically, this is conveniently effected in predominantly acqueoussolvents by reaction of the oligonucleotide with excess reporter groupwhich contains a primary amine in the presence of suitable condensingagents, such as water-soluble carbodiimides, at temperatures in therange of about -20° C. to 50° C. (preferably 20° C. to 40° C.) for 6 to72 hours. Preferred reporter groups of this class include(amino-alkyl)-amino-napthalene-1,2-dicarboxylic acid hydrazides,amino-fluoresceins, aminorhodamines, aminoalkyl luminols,aminoalkylaminobenzenesulfonyl adducts, amino sugars, aminoproteins, andthe like. Furthermore, the chemical synthesis of the initialoligonucleotide product may be accomplished with modified nucleotidemonomers wherein prior to the coupling reaction, such reporter groupsare attached to the linker arm. If any such reporter groups wouldadversely affect the coupling reaction they are appropriately blocked toforestall any such adverse effect. On the other hand, certain otherreporter groups are substantially unreactive with respect to thecoupling reaction and therefore do not require blocking. For example,nitrophenyl adducts may be attached to the substituent group prior tothe coupling reaction, and without masking, may be present on themodified nucleotide monomer during the coupling reaction without adverseeffect.

Reporter groups useful in the method of this invention generally includearomatic, polyaromatic, cyclic, and polycyclic organic moieties whichare further functionalized by inclusion of heteroatoms such as nitrogen,oxygen, sulfur and the like, or proteins capable of producing anappropriately detectable product.

Product oligonucleotides can include more than one type of modificationor more than one modified base. An illustrative example of anoligonucleotide of this type is one of the structure: ##STR13## whereinC^(m) is 5-(3-aminopropyl)cytosine, U^(m) is5-[N-(4-aminobutyl)-1-acrylamido]uracil, and A^(m) is8-[6-2,4-dinitrophenyl)-aminohexyl]aminoadenine. This product is furthermodified by reaction with fluorescein isothiocyanate to provide afluorescein reporter group on C^(m) and U^(m).

Such a product oligonucleotide illustrates the variety of the selectionof modified and unmodified nucleotide units in a product oligonucleotidemade possible by the process of the present invention. Morespecifically, such oligonucleotide illustrates the use of more than onetype of nucleotide unit having its base modified by a linker arm towhich is bound a reporter group whose function may be the same ordifferent from those of reporter groups bound to the substituent groupof other similarly modified nucleotide units thereof. Also illustratedare units whose bases are modified by linker arm to which reportergroups are bound after the coupling reaction, i.e., C^(m) and U^(m),whereas A^(m) is illustrative of a unit whose base is modified by alinker arm to which a dinitrophenyl reporter group was attached prior tothe coupling reaction. Such oligonucleotide additionally illustratesthat it can include more than one nucleotide unit of the same type, andthat it can include units having unmodified bases intermixed with unitshaving modified bases.

Instead of attaching reporter groups to the primary amines of the linkerarm as illustrated in Reaction 6, such amine or other group canalternatively be coupled to suitably activated solid supports. Thisproduces a single strand oligonucleotide which is covalently bound tosuch supports through the modified bases. Such solid supports are usefulin the detection and isolation of complementary nucleic acid components.Alternatively, the modified nucleoside monomers can be coupled to solidsupports prior to the chain extension coupling Reaction 4, to therebyprovide solid supports for such monomers during the coupling reaction.

The following specific examples are provided to enable those skilled inthe art to practice the invention. The examples should not be consideredlimitations upon the scope of the invention, but merely as beingillustrative and representative thereof. To aid in structuralclarification, references are made to the reactions illustrated in theaforementioned process diagram.

EXAMPLE I

This example illustrates the synthesis of a modified nucleosideprecursor 5-(3-trifluoracetylaminopropenyl)-2'-deoxyuridine.

5-Chloromercuri-2'-deoxyuridine (3.6 g, 7.8 mmol) is suspended in 200 mlmethanol. N-Allyltrifluoroacetamide (6.8 ml, 55 mmol) is added, followedby addition of 41 ml of 0.2N lithium tetrachloropalladate in methanol.After 18 hours stirring at room temperature, the reaction is gravityfiltered to remove the black solid palladium, and the yellow methanolicfiltrate is treated with five 200 mg portions of sodium borohydride,then concentrated under reduced pressure to solid residue. The residueis purified by flash column chromatography on silica gel eluting 15 vol% methanol in chloroform. Appropriately pure fractions of product arecombined and concentrated under reduced pressure to give crystalline5-(3-trifluoroacetylamino-propenyl)-2'-deoxyuridine (2.4 g). UV λ_(max)291 nm (ε 7800), λ_(min) 266 nm, (ε 4400); TLC (silica eluting 15 vol %methanol in chloroform) R_(f) =0.4.

EXAMPLE II

This example illustrates the synthesis of a modified nucleosideprecursor5-[N-(trifluoroacetylaminoheptyl)-1-acrylamido]-2'-deoxyuridine.

5-Chloromercuri-2'-deoxyuridine (3.6 g, 7.8 mmol) is suspended in 200 mlmethanol. N-(7-trifluoroacetylaminoheptyl)-acrylamide (55 mmol) isadded, followed by addition of 41 ml of 0.2N lithiumtetrachloropalladate in methanol. After 18 hours stirring at roomtemperature, the reaction is gravity filtered to remove the black solidpalladium, and the yellow methanolic filtrate is treated with five 200mg portions of sodium borohydride, then concentrated under reducedpressure to solid residue. The residue is purified by flash columnchromatography on silica gel eluting 10 vol % methanol in chloroform.Appropriately pure fractions of product are combined and concentratedunder reduced pressure to give crystalline5-[N-(7-trifluoroacetylaminoheptyl)-1-acrylamido]-2'-deoxyuridine (2.8g). UV λ_(max) 302 nm (ε 18000), λ_(min) 230 nm, 280 nm; TLC (silicaeluting 15 vol % methanol in chloroform) R_(f) =0.3.

EXAMPLE III

This example illustrates masking of 5'-hydroxyl to produce5'-dimethoxytrityl-5-(3-trifluoroacetylaminopropenyl)-2'-deoxyuridine asillustrated in Reaction 2.

5-(3-trifluoroacetylaminopropenyl)-2'-deoxyuridine (2.4 g) is thoroughlyevaporated twice from pyridine, then stirred in 40 ml pyridine.Dimethoxytrityl(DMT)chloride (2.3 g, 6.6 mmol) is added, and the mixturestirred at room temperature for four hours. After thin layerchromatography (TLC) on silica eluting 10 vol % methanol in chloroformindicates reaction is complete, the reaction is concentrated to a solidresidue. This residue is purified by column chromatography on silicaeluting chloroform until all faster running impurities have eluted, thenbringing off product with 5 vol % methanol in chloroform. The residue isthen concentrated to give5'-dimethoxytrityl-5-(3-trifluoroacetylaminopropen-1-yl)-2'-deoxyuridineas a white fluffy solid (4 g). Product decomposes upon heating; UVλ_(max) 291 nm, λ_(min) 266 nm; TLC R_(f) 0.6 on silica eluting 10 vol %methanol in chloroform.

EXAMPLE IV

This example illustrates hydrogenation of exocyclic double bond and5'-hydroxyl masking to produce5'-dimethoxytrityl-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine.

Repeating the nucleoside precursor synthesis and 5'-hydroxyl maskingprocedures of Examples I and III, but, prior to the addition of the DMTchloride, subjecting the purified5-(3-trifluoroacetylaminopropenyl)-2'-deoxyuridine to two atmospheres ofhydrogen while stirring at room temperature in methanol over 10%palladium-on-carbon catalyst is productive of5'-dimethoxytrityl-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine.

Examples V to VII illustrate the synthesis of additional modified uracilnucleosides, and subsequent masking of 5'-hydroxyls as represented byReaction 2.

EXAMPLE V

Repeating the nucleoside precursor synthesis and 5' hydroxyl maskingprocedures of Examples I and III, but replacingN-allyltrifluoroacetamide with compounds numbered 1 through 8 below isproductive of the compounds numbers 1' through 8' below, respectively;i.e., substitution of

1 N-(3-butenyl)trichloroacetamide

2 N-(5-hexenyl)trifluoracetamide

3 N-(2-methyl-2-propenyl)trifluoroacetamide

4 N-(4-ethenylphenylmethyl)trifluoroacetamide

5 N-(1-methyl-3-butenyl)trifluoroacetamide

6 N-(12-trichloroaminododecyl)acrylamide

7 N-(pertrifluoroacetylpolylysyl)acrylamide

8 N-(3-trifluoroacetylamidopropyl)acrylamide

is productive of

1'5'-dimethoxytrityl-5-(4-trichloroacetylaminobuten-1-yl)-2'-deoxyuridine

2'5'-dimethoxytrityl-5-(6-trifluoroacetylaminohexen-1-yl)-2'-deoxyuridine

3'5'-dimethoxytrityl-5-(3-trifluoroacetylamino-2-methylpropen-1-yl)-2'-deoxyuridine

4'5'-dimethoxytrityl-5-[2-(4-trifluoroacetylaminomethylphenyl)ethen-1-yl]-2'-deoxyuridine

5'5'-dimethoxyltrityl-5-(4-trifluoroacetylamino-4-methylbuten-1-yl)-2'-deoxyuridine

6'5'-dimethoxytrityl-5-[N-(12-trichloroacetylaminododecyl)-1-acrylamido]-2'-deoxyuridine

7'5'-dimethoxytrityl-5-[N-(pertrifluoroacetylpolylysyl)-1-acrylamido]-2'-deoxyuridine

8'5'-dimethoxytrityl-5-[N-(3-trichloroacetylaminopropyl)-acrylamido]-2'-deoxyuridine

EXAMPLE VI

Repeating the 5' hydroxyl masking procedure of Example III but replacing5-(3-trifluoroacetylaminopropenyl)-2'-deoxyuridine with the5-substituted-2'-deoxyuridines numbered 9 through 18 below is productiveof the products numbered 9' through 18' below, respectively; i.e.substituting

9 5-(propen-1-yl)-2'-deoxyuridine

10 5-(carbmethoxyethyl)-2'-deoxyuridine

11 5-(3-carbmethoxyprop-1-yl)-2'-deoxyuridine

12 5-(4-carbmethoxy-2-methylbuten-1-yl)-2'-deoxyuridine

13 5-(3-cyanopropen-1-yl)-2'-deoxyuridine

14 5-(4-cyano-2-methylbuten-1-yl)-2'-deoxyuridine

15 5-[2-(4-carbmethoxyphenyl)ethen-1-yl]-2'-deoxyuridine

16 5-(4-acetoxybuten-1-yl)-2'-deoxyuridine

17 5-(4-acetoxybut-1-yl)-2'-deoxyuridine

18 5-[4-(2,4-dinitrophenyl)butyl]-2'-deoxyuridine

is productive of the following 5'-dimethoxytrityl-5alkyl-2'-deoxyuridines

9' 5'-dimethoxytrityl-5-(propen-1-yl)-2'-deoxyuridine

10' 5'-dimethoxytrityl-5-(2-carbmethoxyethyl)-2'-deoxyuridine

11' 5'-dimethoxytrityl-5-(3-carbmethoxyprop-1-yl)-2'-deoxyuridine

12'5'-dimethoxytrityl-5-(4-carbmethoxy-2-methylbuten-1-yl)-2'-deoxyuridine

13' 5'-dimethoxytrityl-5-(3-cyanopropen-1-yl)-2'-deoxyuridine

14' 5'-dimethoxytrityl-5-(4-cyano-2-methylbuten-1-yl)-2'-deoxyuridine

15'5'-dimethoxytrityl-5-[2-(4-carbmethoxyphenyl)ethen-1-yl]-2'-deoxyuridine

16' 5'-dimethoxytrityl-5-(4-acetoxybuten-1-yl)-2'-deoxyuridine

17' 5'-dimethoxytrityl-5-(4-acetoxybut-1-yl)-2'-deoxyuridine

18' 5'-dimethoxytrityl-5-[4-(2,4-dinitrophenyl)butyl]-2'-deoxyuridine

EXAMPLE VII

Repeating the nucleoside precursor synthesis and 5'-hydroxyl maskingprocedures of Examples I-VI, but replacing5-chloromercuri-2'-deoxyuridine with 5-chloromercuriuridine isproductive of the corresponding 5'-dimethoxytrityl-5-substituteduridines.

Examples VIII to XI illustrate the synthesis of modified cytosinenucleosides. Since cytosine nucleosides, as well as adenosinenucleosides, have reactive groups in their bases unlike the uracilnucleosides, such reactive groups are masked to prevent unwantedreactions therewith. These examples illustrate masking of reactivegroups on the cytosine base moiety as in Reaction 1, as well as maskingof the 5'-hydroxyl as in Reaction 2.

EXAMPLE VIII 5-(3-trifluoroacetylaminopropenyl)-N⁴-benzoyl-2'-deoxycytidine

Repeating the nucleoside precursor synthesis procedure of Example I, butreplacing 5-chloromercuri-2'-deoxyuridine with5-chloromercuri-2'-deoxycytidine is productive of5-(3-trifluoroacetylaminopropenyl)-2'-deoxycytidine (UV_(max) 287 mn).Purified 5-(3-trifluoroacetyl-aminopropenyl)-2'-deoxycytidine (1.3 g,4.6 mmol) is stirred in 80 ml anhydrous ethanol, benzoyl anhydride (1.5g, 7 mmol) is added, and the reaction refluxed. Five additional 1.5 gportions of benzoyl anhydride are added hourly. After the reaction isjudged complete by thin layer chromatography [silica plates elutingn-butanol/methanol/conc NH₄ OH/H₂ O (60:20:1:20)in 6-10 hours, thereaction is cooled and concentrated under reduced pressure to asemisolid. The solid is triturated with ether three times, decanted anddried. The crude product is crystallized from water to givechromatographically pureN4-benzoyl-5-(3-trifluoroacetylaminopropenyl)-2'-deoxycytidine as awhite solid. The product decomposes above 120° C.; UV λ_(max) 311 nm.

EXAMPLE IX 5' Dimethoxytrityl-5-(3-trifluoroacetylaminopropenyl)-N⁴-benzoyl-2'-deoxycytidine

Repeating the 5'-hydroxyl masking procedure of Example III, butreplacing 5-(3-trifluoroacetylaminopropenyl)-2'-deoxyuridine with5-(3-trifluoroacetyleaminopropenyl)-N⁴ -benzoyl-2'-deoxycytidine isproductive of 5'-dimethoxytrityl-5-(3-trifluoroacetylaminopropenyl)-N⁴-benzoyl-2'-deoxycytidine.

EXAMPLE X

Repeating the nucleoside precursor synthesis and 5'hydroxyl maskingprocedures of Examples VIII and IX, but replacingN-allyltrifluoroacetamide with the respective N-alkyltrifluoroacetamidesof Example V is productive of the corresponding5'-dimethoxytrityl-5-(trifluoroacetylaminoalkyl)-N⁴-benzoyl-2'-deoxycytidines numbered 1" through 8" below.

1" 5'-dimethoxytrityl-5-(4-trifluoroacetylaminobuten-1-yl)-N⁴-benzoyl-2'-deoxycytidine

2" 5'-dimethoxytrityl-5-(6-trifluoroacetylaminohexen-1-yl)-N⁴-benzoyl-2'-deoxycytidine

3" 5'-dimethoxytrityl-5-(3-trifluoroacetylamino-2methylpropen-1-yl)-N⁴-benzoyl-2'-deoxycytidine

4"5'-dimethoxytrityl-5-[2-(4trifluoroacetylaminomethylphenyl)ethen-1-yl]-N.sup.4-benzoyl-2'-deoxycytidine

5" 5'-dimethoxytrityl-5-(4-trifluoroacetylamino-4-methylbuten-1-yl)-N⁴-benzoyl-2'-deoxycytidine

6"5'-dimethoxytrityl-5-[N-(12-trifluoroacetylaminododecyl)-1-acrylamido]-N.sup.4-benzoyl-2'-deoxycytidine

7"5'-dimethoxytrityl-5-[N-(pertrifluoroacetylpolylysyl)-1-acrylamido]-N.sup.4-benzoyl-2'-deoxycytidine

8"5'-dimethoxytrityl-5-[N-(3-trifluoroacetylaminopropyl)-1-acrylamido]-N.sup.4-benzoyl-2'-deoxycytidine

EXAMPLE XI Synthesis of 5'-dimethoxytrityl-N⁴-benzoyl-5-(2-carbmethoxyethenyl)-2'-deoxycytidine

5-(2-Carbmethoxyethenyl)-2'-deoxycytidine (0.82 g, 2.6 mmol) is stirredin 50 ml anhydrous ethanol. Benzoic anhydride (500 mg, 2.2 mmol) isadded, and the reaction heated to reflux. Five additional 500 mgportions of benzoic anhydride are added hourly. After the reaction isjudged complete by thin layer chromatography (usually 6-8 hours) thereaction is cooled, and evaporated under reduced pressure to a yellowsemi-solid. Chromatography on silica gel eluting a linear 1:19 to 1:3methanol/chloroform mixture followed by thorough evaporation ofappropriately combined fractions gives N⁴-benzoyl-5-(2-carbmethoxyethenyl)-2'-deoxycytidine as an amorphous whitesolid. UV λ_(max) 296 nm, λ_(min) 270 nm. The solid is dried thoroughly,and dissolved in 20 ml pyridine. Dimethoxytrityl chloride (1.1 eq) isadded, and the reaction stirred at ambient temperature for six hours.Concentration to a solid followed by column chromatography on silica geleluting 10% methanol in chloroform yields 5'-dimethoxytrityl-N⁴-benzoyl-5-(2-carbmethoxyethenyl)-2'-deoxycytidine as a fluffy off-whitesolid.

EXAMPLE XII

Repeating the nucleoside precursor synthesis procedure of Example XI,but replacing 5-(2-carbmethoxy-hetenyl)-2'-deoxycytidine with thefollowing compounds numbered 19 through 27 below is productive of thecorresponding compounds numbered 19' through 27' below, respectively,i.e., substituting:

19 5-(2-carbmethoxyethyl)-2'-deoxycytidine

20 5-(3-carbmethoxyprop-1-yl)-2'-deoxycytidine

21 5-(4-carbmethoxy-2-methylbuten-1-yl)-2'-deoxycytidine

22 5-(3-cyanopropen-1-yl)-2'-deoxycytidine

23 5-(4-cyano-2-methylbuten-1-yl)-2'-deoxycytidine

24 5-[2-(4-carbmethoxyphenyl)ethen-1-yl]-2'-deoxycytidine

25 5-(4-acetoxybuten-1-yl)-2'-deoxycytidine

26 5-(4-acetoxybut-1-yl)-2'-deoxycytidine

27 5-[4-(2,4-dinitrophenyl)butyl]-2'-deoxycytidine

is productive of the following 5'-dimethoxytrityl-N⁴-benzoyl-5-alkyl-2'-deoxycytidines:

19' 5'-DMT-N⁴ -benzoyl-5-(2-carbmethoxyethen-1-yl)-2'-deoxycytidine

20' 5'-DMT-N⁴ -benzoyl-5-(3-carbmethoxyprop-1-yl)-2'-deoxycytidine

21' 5'-DMT-N⁴-benzoyl-5-(4-carmethoxy-2-methylbuten-1-yl)-2'-deoxycytidine

22' 5'-DMT-N⁴ -benzoyl-5-(3-cyanopropen-1-yl)-2'-deoxycytidine

23' 5'-DMT-N⁴ -benzoyl-5-(4-cyano-2-methylbuten-1-yl)-2'-deoxycytidine

24' 5'-DMT-N⁴-benzoyl-5-[2-(4-carbmethoxyphenyl)ethen-1-yl]-2'-deoxycytidine

25' 5'-DMT-N⁴ -benzoyl-5-(4-acetoxybuten-1-yl)-2'-deoxycytidine

26' 5'-DMT-N⁴ -benzoyl-5-(4-acetoxybut-1-yl)-2'-deoxycytidine

27' 5'-DMT-N⁴ -benzoyl-5-[4-(2,4-dinitrophenyl)butyl]-2'-deoxycytidine

Similarly, the use of the other acid anhydrides, e.g., acetic anhydride,anisoyl anhydride, or tolyl anhydride, is productive of thecorresponding N⁴ -acyl or N⁴ -acetyl 5-alkyl-2'-deoxycytidines ofExamples X and XI wherein benzoyl is replaced by acetyl or acyl.

EXAMPLE XIII

Repeating the nucleoside precursor synthesis and 5'-hydroxyl maskingprocedures of Examples VIII to X, but replacing5-chloromercuri-2'-deoxycytidine with 5-chloromercuricytidine isproductive of the corresponding 5'-dimethoxytrityl-N⁴-benzoyl-5-substituted cytidines.

EXAMPLE XIV

This example typifies the masking of reactive base moieties and themasking of 5'-hydroxyl of adenine nucleosides.

N⁶ -benzoyl-8-(6-aminohexyl)amino-2'-deoxyadenosine (4 mmol) is stirredin 60 ml anhydrous ethanol. Trifluoroacetic anhydride (6 mmol) is added,and the reaction stirred at room temperature. Two additional portions oftrifluoroacetic anhydride are added hourly. After four hours, thereaction is concentrated to a solid residue, and lyophilized overnight.The crude N⁶-benzoyl-8-(6-trifluoroacetylaminohexyl)amino-2'-deoxyadenosine is driedthoroughly and concentrated to a solid residue twice from pyridine. Thesolid is stirred in 40 ml of pyridine, and dimethoxytrityl chloride (6.5mmol) is added. After four hours, the reaction is concentrated to leavea solid residue. Purification by column chromatography on silica geleluting a multi-step gradient of 0 to 15% methanol in chloroform gives5'-dimethoxytrityl-N⁶-benzoyl-8-(6-trifluoroacetylaminohexyl)amino-2'-deoxyadenosine as anoff-white solid.

Examples XV and XVII typify the activation of 5'-masked 5-substituted,and naturally occurring nucleosides, to their respectivephosphomonochloridites, as illustrated in Reaction 3 of the diagram.

EXAMPLE XV Preparation of 3'-phosphomonochloridite of5'-DMT-5-(3-trifluoroacetylaminoprop-1-yl)-2'-deoxyuridine

Dry 5'-DMT-5-(3-trifluoroacetylaminoprop-1-yl)-2'-deoxyuridine (1.54 g,2.2 mmol) is lyophilized from 20 ml benzene three times for more thantwelve hours each to remove residual water and solvents. The resultingvery fluffy white powder is transferred to a nitrogen atmosphere whilestill under vacuum and dissolved in anhydrous acetonitrile containing 5vol % 2,6-lutidine to a final nucleoside concentration of 30 mM. Whileswirling vigorously under nitrogen, one rapid bolus ofmethylphosphodichloridite (1.0 eq) is added by syringe. The reaction isswirled for about one minute under nitrogen. The resulting crude5'-DMT-5-(3-trifluoroacetylaminoprop-1-yl)-2'-deoxyuridine3'-methylphosphomonochloridite reaction solution is then used directlyfor deoxyoligonucleotide synthesis (Example XVIII) with no furtherpurification, [³¹ P-NMR(CH₃ CN/CDCl₃) generally indicates 40-70 mol %desired product (167.5 ppm); remainder is composed ofbis-3',3'-[5'DMT-5-(3-trifluoroacetylaminoprop-1-yl)-2'-deoxyuridylyl]methylphosphite(140 ppm) and 5'-DMT-5-(3-trifluoroacetylaminoprop-1-yl)-2'-deoxyuridine3'-methylphosphonate (9.5 ppm), the latter product being formed inamounts reflecting the presence of water in the reaction.]

EXAMPLE XVI Preparation of 3'-phosphomonochloridites of the naturallyoccurring 2'-deoxynucleosides

Repeating the procedure of Example XV, but replacing5'-DMT-5-(3-trifluroacetylaminoprop-1-yl)-2'-deoxyuridine with:

5'-DMT-2'-deoxythymidine

5'-DMT-N⁴ -benzoyl-2'-deoxycytidine

5'-DMT-N⁶ -benzoyl-2'-deoxyadenosine

5'-DMT-N² -isobutyryl-2'-deoxyguanosine

is productive of the corresponding phosphomonochloridites, viz.:

5'-DMT-2'-deoxythymidine 3'-methylphosphomonochloridite

5'-DMT-N⁴ -benzoyl-2'-deoxycytidine 3'-methylphosphomonochloridite

5'-DMT-N⁶ -benzoyl-2'-deoxyadenosine 3'-methylphosphomonochloridite

5'-DMT-N² -isobutyryl-2'-deoxyguanosine 3'-methylphosphomonochloridite

EXAMPLE XVII

Repeating the phosphomonochloridite synthesis procedures of Examples XVand XVI, but replacing methylphosphodichloridite witho-chlorophenylphosphodichloridite is productive of the corresponding5'-DMT-nucleoside 3'-phosphomonochloridites, viz.:

5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine

3'- o-chlorophenylphosphomonochloridite

5'-DMT-2'-deoxythymidine 3'-o-chlorophenylphosphomonochloridite

5'-DMT-N⁴ -benzoyl-2'-deoxycytidine 3'-o-chlorophenylphosphomonochloridite

5'-DMT-N⁶ -benzoyl-2'-deoxyadenosine 3'-o-chlorophenylphosphomonochloridite

5'-DMT-N² -isobutyryl-2'-deoxyguanosine 3'-o-chlorophenylphosphomonochloridite

Similarly, the use of p-chlorophenylphosphodichloridite is productive ofthe analogous 3'-p-chlorophenylphosphomonochloridite adducts. [³² P]NMR(CH₃ CNDCl₃) of o-chlorophenylphosphomonochloridite products 160.7,160.5 ppm (diasteriomers).

Examples XVIII-XXIV typify the chemical synthesis of oligonucleotideswhich incorporate modified bases, as illustrated by Reactions 4 and 5 inthe diagram.

EXAMPLE XVIII Synthesis of deoxyoligonucleotides containing5-(3-aminopropyl)-uracil and naturally occurring nucleotide units

The phosphomonchloridite synthesis procedures of Examples XV and XVI areaccomplished immediately before deoxyoligonucleotide synthesis, and theresulting products are used directly as 30 mM crude3'-methylphosphomonochloridites in anhydrous acetonitrile/5 vol %2,6-lutidine.

Solid support (5-DMT-N⁶ -benzoyl-2'-deoxyadenosine 3'-succinamidepropylsilica, 250 mg, 20 μeq) is put into a suitable reaction flow vessel(glass or Teflon® column or funnel). The solid support is preconditionedby successive treatments with acetonitrile/5 vol % lutidine, 2 w/v %iodine in tetrahydrofuran/water/lutidine for 2 minutes, acetonitrile/5%lutidine, chloroform, 4 vol % dichloroacetic acid in chloroform for 2.5minutes, and acetonitrile/5% lutidine, where treatments are totalvolumes of 5-15 ml in either 2 or 3 portions or by constant flow, asdesired.

The deoxyoligonucleotide is synthesized in accordance with Reaction 4 bysequential addition of the desired activated 5'-DMT-nucleoside3'-methylphosphomonochloridite monomer and coupling thereof to the free5'-hydroxyl of the terminal unit of the growing nucleotide chain, whichunit is initially the only unit of the chain, i.e., the deoxyadenosinebased unit comprising the solid support. Additions are by reaction of 10ml of the crude 30 mM monochloridites chosen from Examples XV and XVIwith the now unprotected 5'-hydroxyl of the chain in either 2 or 3portions or by constant flow, for 2 to 6 minutes. The firstphosphomonochloridite addition followed by one complete reagent cycleconsists of sequential treatments with:

5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine3'-methylphosphomonochloridite

acetronitrile/lutidine wash

capping for 5 minutes with 0.3M 4-dimethylaminopyridine in aceticanhydride/lutidine/tetrahydrofuran (1:3:2)

acetonitrile/5% lutidine wash

oxidation with 2% iodine in tetrahydrofuran/water/lutidine (6:2:1) for 2minutes

acetronitrile/5% lutidine wash

chloroform wash

removal of DMT by 2.5 minute treatment with 4 vol % dichloroacetic acidin chloroform

chloroform wash

acetonitrile/lutidine wash

The above cycle is repeated thirteen times, each time replacing5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine3'-methylphosphomonochloridite with a different one of the following3'-methylphosphomonochloridites:

5'-DMT-2'-deoxythymidine 3'-methylphosphomonochloridite

5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine3'-methylphosphomonochloridite

5'-DMT-N⁶ -benzoyl-2'-deoxyadenosine 3'-methylphosphomonochloridite

5'-DMT-N⁴ -benzoyl-2'-deoxycytidine 3'-methylphosphomonochloridite

5'-DMT-N² -isobutyryl-2'-deoxyguanosine 3'-methylphosphomonochloridite

5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine3'-methylphosphomonochloridite

5'-DMT-2'-deoxythymidine 3'-methylphosphomonochloridite

5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine3'-methylphosphomonochloridite

5'-DMT-deoxythymidine 3'-methylphosphomonochloridite

5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine3'-methylphosphomonochloridite

5'-DMT-N² -isobutyryl-2'-deoxyguanosine 3'-methylphosphomonochloridite

5'-DMT-N⁶ -benzoyl-2'-deoxyadenosine 3'-methylphosphomonochloridite

5'-DMT-N⁴ -benzoyl-2'-deoxycytidine 3'-methylphosphomonochloridite

in respective order, deleting dichloroacetic acid treatment during thelast reagent cycle. The support is transferred and treated with 2 mlconcentrated ammonium hydroxide for 4 hours at ambient temperature torelease the product from the support. The supernatant is removed, thesolid washed three times with 0.5 ml concentrated ammonium hydroxide,and the combined supernatants are sealed and heated at 50° C. overnight.The clear yellow supernatant is lyophilized thoroughly. Initialpurification is accomplished by reverse phase high pressure liquidchromatography (HPLC) on an RP-8 (C-8) column eluting a 60 minute lineargradient of 0 to 30% vol % acetonitrile in 25 mM ammonium acetate, pH6.8. The 5'-DMT-terminated product, eluting as a sharp peak at about 40minutes, is collected; all shorter chains, both capped and uncapped,elute before 25 minutes. The collected product is evaporated to a solidresidue, treated with 80% acetic acid at ambient temperature for 20minutes (to remove DMT), lyophilized to a solid residue, and dissolvedin a small amount of aqueous buffer. The product, generally greater than90% homogeneous after HPLC, is further purified by conventionalelectrophoresis on 20% polyacrylamide gels (1 to 6 mm thick) by excisionand extraction of the appropriate product band (product generallymigrates slower than unmodified deoxyligonucleodites of similar length).The purified 5-aminopropyl-uracil-containingpentadecadeoxyoligonucleotide product illustrated diagrammaticallybelow, wherein U^(m) =5-(3 aminopropyl)uracil, is thereby produced.##STR14##

Note the conventional deprotection of the oligonucleotide with ammoniahas also removed the trifluoroacetyl masking group on the substituent.

The length and sequence of this oligonucleotide may then be determinedby ³² P-kinasing and sequencing using suitable protocols, for examplethe protocols heretofore used to determine the length and sequence ofthe prior art oligonucleotides in which the bases of the nucleotideunits therein are unmodified.

Similarly, intentional variation of the order and number ofmethylphosphomonochloridite additions employed here is productive ofother 5-(modified)uracil-containing deoxyoligonucleotides which vary inselected length and base sequence. In addition, replacement of thenucleoside 3'-methylphosphomonchloridite adducts of Examples XV and XVIwith the corresponding 3'-o- or p-chloro-phenylphosphomonochloriditeadducts of Example XVII and inclusion of pyridinium oximate treatment toremove chlorophenyl blocking groups (at the end of thedeoxyoligonucleotide synthesis and before concentrated ammoniumhydroxide treatment) is productive of the same deoxyoligonucleotideproducts.

EXAMPLE XIX

Repeating the phosphomonochloridite and deoxyoligonucleotide synthesisprocedures of Examples XV to XVIII, but replacing5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine with the5'-DMT-5alkyl-2'-deoxy-uridines numbered 28 through 38 below isproductive of the corresponding oligonucleotides having uracil basesU^(m) numbered 28' through 38' below, respectively, i.e., substituting:

28 5'-dimethoxytrityl-5-(3-trifluoroacetylaminopropen-1-yl)-deoxyuridine

29 5'-dimethoxytrityl-5-(4-trifluoroacetylaminobut-1-yl)-2'-deoxyuridine

305'-dimethoxytrityl-5-(4-trifluoroacetylaminobuten-1-yl)-2'-deoxyuridine

31 5'-dimethoxytrityl-5-(6-trifluoroacetylaminohex-1-yl)-2'-deoxyuridine

325'-dimethoxytrityl-5-(6-trifluoroacetylaminohexen-1-yl)-2'-deoxyuridine

335'-dimethoxytrityl-5-(2-trifluoroacetylaminoprop-2-yl)-2'-deoxyuridine

345'-dimethoxytrityl-5-(3-trifluoroacetylamino-2-methyl-propen-1-yl)-2'-deoxyuridine

355'-dimethoxytrityl-5-(3-trifluoroacetylamino-2-methyl-prop-1-yl)-2'-deoxyuridine

365'-dimethoxytrityl-5-[2-(4-trichloroacetylaminomethylphenyl)ethen-1-yl]-2'-deoxyuridine

375'-dimethoxytrityl-5-[N-(pertrifluoroacetylpolylysyl)-1-acrylamido]-2'-deoxyuridine

385'-DMT-5-[N-(7-trifluoro-acetylaminoheptyl)-1-acrylamido]-2'-deoxyuridine

is productive of the deoxynucleotides corresponding to the product ofExample XVIII, wherein U^(m) is:

28' 5-(3-aminopropen-1-yl)uracil

29' 5-(4-aminobut-1-yl)uracil

30' 5-(4-aminobuten-1-yl)uracil

31' 5-(6-aminohex-1-yl)uracil

32' 5-(6-aminohexen-1-yl)uracil

33' 5-(3-aminoprop-2-yl)uracil

34' 5-(3-amino-2-methylpropen-1-yl)uracil

35' 5-(3-amino-2-methylprop-1-yl)uracil

36' 5-[2-(4-aminoethylphenyl)ethen-1-yl]uracil

37' 5-[N-(polylysyl)-1-acrylamido]uracil

38' 5-[N-(7-aminoheptyl)-1-acrylamido]uracil

Similarly, by employing other5'-DMT-5-(acylaminoalkyl)-2'-deoxyuridines, the analogousdeoxyoligonucleotides are produced.

EXAMPLE XX

Repeating the phosphomonochloridite and deoxyolgionucleotide synthesisprocedures of Examples XV to XVIII, but replacing5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine with5-substituted-2'-deoxyuridines numbered 37a through 46 below isproductive of the corresponding oligonucleotides having the U^(m) uracilbases numbered 37a' through 46' below respectively; i.e., substituting:

37a 5'-DMT-5-(propen-1-yl)-2'-deoxyuridine

38a 5'-DMT-5-(2-carbmethoxyethyl)-2'-deoxyuridine

39 5'-DMT-5-(3-carbmethoxyprop-1-yl)-2'-deoxyuridine

40 5'-DMT-5-(4-carbmethoxy-2-methylbuten-1-yl)-2'-deoxyuridine

41 5'-DMT-5-(3-cyanopropen-1-yl)-2'-deoxyuridine

42 5'-DMT-5-(4-cyano-2-methylbuten-1-yl)-2'-deoxyuridine

43 5'-DMT-5-[2-(4-carbmethoxyphenyl)ethen-1-yl]-2'-deoxyuridine

44 5'-DMT-5-(4-acetoxybuten-1-yl)-2'-deoxyuridine

45 5'-DMT-5-(4-acetoxybut-1-yl)-2'-deoxyuridine

46 5'-DMT-5-[4-(2,4-dinitrophenyl)butyl]-2'-deoxyuridine

productive of the products wherein, U^(m) is:

37a' 5-(propen-1-yl)uracil

38a' 5-(2-carboxyethyl)uracil

39' 5-(3-carboxyprop-1-yl)uracil

40' 5-(4-carboxy-2-methylbuten-1-yl)uracil

41' 5-(3-cyanopropen-1-yl)uracil

42' 5-(4-cyano-2-methylbuten-1-yl)uracil

43' 5-[2-(4-carboxyphenyl)ethen-1-yl]uracil

44' 5-(4-hydroxybuten-1-yl)uracil

45' 5-(4-hydroxybut-1-yl)uracil

46' 5-[4-(2,4-dinitrophenyl)butyl]uracil

Note: In 46' the dinitrophenyl is a ligand recognition type reportergroup, i.e. use of antidinitrophenyl antibody as the ligand. Similarly,by employing other appropriate 5'-DMT-5-alkyl-2'-deoxyuridines, theanalogous deoxyoligonucleotides are produced.

EXAMPLE XXI

Repeating the procedures of Examples XV to XVIII, but replacing5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine with 5'-DMT-N⁴-benzoyl-5-(3-trichloroacetylaminopropyl)-2'-deoxycytidine is productiveof deoxyoligonucleotides as in Example XVIII wherein U^(m)[5-(3-aminopropyl)-uracil] is replaced by 5-(3-aminopropyl)cytosines.For example, ##STR15## where C^(m) =5-(3-aminopropyl)cytosine.

EXAMPLE XXII

Repeating the deoxyoligonucleotide synthesis procedure of Example XXIbut replacing 5'-DMT-N⁴-5-(3-trichloroacetylaminopropyl)-2'-deoxycytidine with compoundsnumbered 47 through 57 below is productive of the correspondingoligonucleotides having the C^(m) cytosine bases numbered 47' through57' below, respectively, i.e., substituting:

47 5-DMT-N⁴-benzoyl-5-(3-trifluoroacetylaminopropen-1-yl)-2'-deoxycytidine

48 5'-DMT-N⁴-benzoyl-5-(4-trifluoroacetylaminobut-1-yl)-2'-deoxycytidine

49 5'-DMT-N⁴-benzoyl-5-(4-trifluoroacetylaminobuten-1-yl)-2'-deoxycytidine

50 5'-DMT-N⁴-benzoyl-5-(6-trifluoroacetylaminohex-1-yl)-2'-deoxycytidine

51 5'-DMT-N⁴-benzoyl-5-(6-trifluoroacetylaminohexen-1-yl)-2'-deoxycytidine

52 5'-DMT-N⁴-benzoyl-5-(3-trifluoroacetylaminoprop-2-yl)-2'-deoxycytidine

53 5'-DMT-N⁴-benzoyl-5-(3-trifluoroacetylamino-2-methylpropen-1-yl)-2'-deoxycytidine

54 5'-DMT-N⁴-benzoyl-5-(3-trifluoroacetylamino-2-methylprop-1-yl)-2'-deoxycytidine

55 5'-DMT-N⁴-benzoyl-5[2-(4-trifluoroacetylaminomethylphenyl)ethen-1-yl]-2'-deoxycytidine

56 5'-DMT-N⁴-benzoyl-5-[N-(pertrifluoroacetylpolylysyl)-1-acrylamido)-2'-deoxycytidine

57 5'-DMT-N⁴-benzoyl-5-[N-(trifluoroacetylaminoheptyl)acrylamido]-2'-deoxycytidine

productive of the products wherein C^(m) is:

47' 5-(3-aminopropen-1-yl)cytosine

48' 5-(4-aminobut-1-yl)cytosine

49' 5-(4-aminobuten-1-yl)cytosine

50' 5-(6-aminohex-1-yl)cytosine

51' 5-(6-aminohexen-1-yl)cytosine

52' 5-(3-aminoprop-2-yl)cytosine

53' 5-(3-amino-2-methylpropen-1-yl)cytosine

54' 5-(3-amino-2-methylprop-1-yl)cytosine

55' 5-[2-(4-aminomethylphenyl)ethen-1-yl]cytosine

56' 5-[N-(polylysyl)-1-acrylamido]cytosine

57' 5-[N-(7-aminoheptyl)-1-acrylamido]cytosine

Similarly, by employing other N⁴-acyl-5-(acylaminoalkyl)-2'-deoxycytidines the analogousdeoxyoligonucleotides are produced.

EXAMPLE XXIII

Repeating the deoxyoligonucleotide synthesis procedure of Example XXI,but replacing 5'-DMT-N⁴-benzoyl-5-(3-trifluoroacetylaminopropyl)-2'-deoxycytidine with thecompounds numbered 58 through 68 below is productive of thecorresponding oligonucleotides having the C^(m) cytosine bases numbered58' through 68' below, respectively, i.e., substituting:

58 5'-DMT-N⁴ -benzoyl-5-(propen-1-yl)-2'-deoxycytidine

59 5'-DMT-N⁴ -benzoyl-5-(2-carbmethoxyethyl)-2'-deoxycytidine

60 5'-DMT-N⁴ -benzoyl-5-(2-carbmethoxyethen-1-yl)-2'-deoxycytidine

61 5'-DMT-N⁴ -benzoyl-5-(3-carbmethoxyprop-1-yl)-2'-deoxycytidine

62 5'-DMT-N⁴-benzoyl-5-(4-carbmethoxy-2-methylbuten-1-yl)-2'-deoxycytidine

63 5'-DMT-N⁴ -benzoyl-5-(3-cyanopropen-1-yl)-2'-deoxycytidine

64 5'-DMT-N⁴ -benzoyl-5-(4-cyano-2-methylbuten-1-yl)-2'-deoxycytidine

65 5'-DMT-N⁴-benzoyl-5-[2-(4-carbmethoxyphenyl)ethen-1-yl]-2'-deoxycytidine

66 5'-DMT-N⁴ -benzoyl-5-(4-acetoxybuten-1-yl)-2'-deoxycytidine

67 5'-DMT-N⁴ -benzoyl-5-(4-acetoxyabut-1-yl)-2'-deoxycytidine

68 5'-DMT-N⁴ -benzoyl-5-[4-(2,4-dinitrophenyl)butyl]-2-deoxycytidine

is productive of the products wherein C^(m) is:

58' 5-(propen-1-yl)cytosine

59' 5-(2-carboxyethyl)cytosine

60' 5-(2-carboxyethen-1-yl)cytosine

61' 5-(3-carboxyprop-1-yl)cytosine

62' 5-(4-carboxy-2-methylbuten-1-yl)cytosine

63' 5-(3-cyanopropen-1-yl)cytosine

64' 5-(4-cyano-2-methylbuten-1-yl)cytosine

65' 5-[2-(4-carboxyphenyl)ethen-1-yl]cytosine

66' 5-(4-hydroxybuten-1-yl)cytosine

67' 5-(4-hydroxybut-1-yl)cytosine

68' 5-[4-(2,4-dinitrophenyl)butyl]cytosine

Similarly, by employing other appropriate 5'-DMT-N⁴-acyl-5-alkyl-2'-deoxycytidines the analogous deoxyoligonucleotides areproduced.

EXAMPLE XXIV

Repeating the phosphomonochloridite and deoxyoligonucleotide synthesisprocedures of Examples XV-XXIII, but replacing5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine with 5'-DMT-N⁶-benzoyl-8-(6-trifluoroacetylaminohexyl)amino-2'-deoxyadenosine isproductive of deoxyoligonucleotides as in Example XVIII, except that theU^(m) is replaced by A^(m), and A^(m)=8-(6-aminohexyl)amino-2'-deoxyadenosine.

Examples XXVI to XXIX typify the binding of reporter groups tooligonucleotides containing appropriately modified bases, as illustratedin Reaction 6.

EXAMPLE XXV Fluoresceinated deoxyoligonucleotides

A purified pentadeca-nucleotide (from Example XVIII) of the structure##STR16## [where U^(m) is 5-[N-(7-aminoheptyl)-1-acrylamido]uracil isdissolved at 25 A₂₆₀ units per ml in aqueous 300 mM sodium borate orsodium carbonate buffer, pH 9.5, containing 30 mM sodium chloride. Solidfluorescein isothiocyanate (0.5 mg per ml) is added, and the mixturesealed and shaken gently at 4° C. to 25° C. overnight. the reaction ischromatographed directly on a column of G-50 Sephadex® to separateunbound fluorescein adducts which are retained; the fluoresceinateddeoxyoligonucleotide adducts elute near the void volume. Early fractionscontaining significant A₂₆₀ units are combined and lyophilized to solidproduct of structure similar to the startingpetadecadeoxyoligonucleotide where U^(m) is now either ##STR17## orunreacted 5-[N-(7-aminoheptyl)-1-acrylamido]uracil. λ_(max) (H₂ O) 262nm, 498 nm.

Repeating the procedure on compounds recited in Examples XIX, XXI, XXII,and XXIV is productive of the corresponding fluoresceinated orpolyfluoresceinated deoxyoligonucleotides in like manner.

EXAMPLE XXVI

Attachment of reporter groups other than fluorescein can be accomplishedby repeating the procedure of Example XXV, but replacing fluoresceinisothiocyanate with, for example:

2,4-dinitrophenyl isothiocyanate

1-fluoro-2,4-dinitrobenzene

aminoethyl isoluminol isothiocyanate

aminoethylaminonaphthalene-1,2-carboxylic hydrazide isothiocyanate

N,N'-bis(alkylsulfonyl)-N-aryl-N'-isothiocyanatoaryl-dioxamide

m-sulfonyl aniline isothiocyanate

N-hydroxysuccinimidyl biotin

9-(n-hydroxysuccinimidyl carboxy)-N-methylacridine,

or cyanogenbromide-activated Sepharose® is productive of thecorresponding adducts wherein the attached group is other thanfluorescein.

EXAMPLE XXVII Attachment of isoluminol and free primary amine-containingreporter groups

A purified pentadecanucleotide from Example XVIII of the structure:##STR18## [where U^(m) is 5-(2-carboxyethenyl)uracil] is dissolved inwater at 30 A₂₆₀ units per ml, and diluted with one volume pyridine.Aminobutyl ethyl isoluminol is added to a final concentration of 1mg/ml, followed by addition of a five-fold molar excess of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The reaction is sealedand shaken gently in the dark for 12 to 48 hours. The reaction mixtureis concentrated under reduced pressure to a solid residue, andchromatographed directly on a column of G-50 Sephadex®; theisoluminol-deoxyoligonucleotide conjugates elute near the void volume.Early fractions containing siginficant A₂₆₀ units are combined andlyophilized to solid product of structure similar to the startingdeoxyoligonucleotide where U^(m) is now either: ##STR19## or unreacted5-(2-carboxyethenyl)uracil.

Repeating the procedure on compounds from Examples XVI and XXIV whereinR₂ contains carboxy is productive of the correspondingdeoxyoligonucleotide-isoluminol adducts in like manner.

Repeating the procedure, but replacing aminobutyl isoluminol with otherreporter groups containing a free primary amine is productive of thecorresponding deoxyoligonucleotide-reporter adducts in like manner.

EXAMPLE XXVIII Attachment Of Dinitrophenyl Reporter Groups

A purified nanonucleotide of the structure: ##STR20## where A^(m)=8-(6-aminohexyl)aminoadenine is dissolved at 20 A₂₆₀ units per ml in250 mM sodium carbonate buffer, pH 9, and 1-fluoro-2,4-dinitrobenzene isadded. The reaction solution is shaken at ambient temperature overnight,then chromatographed directly on a column of Sephadex® G-50. Earlyfractions containing significant A₂₆₀ units ate combined andconcentrated to give an oligonucleotide product similar to the startingdecanucleotide wherein A^(m) is now either: ##STR21## or unreacted8-(6-aminohexyl)aminoadenine.

Repeating the procedure, but replacing 8-(6-aminohexyl)aminoadenine withother modified bases containing a free primary amine, is similarlyproductive of the corresponding dinitrophenylated oligonucleotideadducts.

EXAMPLE XXIX Preparation of5'-DMT-5-[N-trifluoroacetylaminoheptyl)1-acrylamido]-2'-deoxyuridinemethyl N,N-diisopropylphosphoramidite

The method followed was that of Beaucage and Caruthers, (1981). Tet.Lett. 22:1859, with some modification. Dry5'-DMT-5-[N-trifluoroacetylaminoheptyl)1-acrylamido]-2'-deoxyuridine(3.0 g, 3.7 mmol) from Example II was dissolved in 12 ml dichloromethanein a dry 50 ml round bottom flask with a septum cap. The flask wasflushed with argon gas and evacuated several times to remove residualwater vapor. 2.1 ml triethylamine was added with a dry syringe, and themixture cooled to 0° C. While stirring, 1.44 mlN,N-diisopropyl-methylphosphoramidic chloride was added drop-wise with asyringe. The mixture was allowed to warm to room temperature and stirred30 minutes. 20 ml ethyl acetate was added, the contents were mixed, andextracted with 50 ml cold aqueous NaCl (saturated). The aqueous phasewas re-extracted two times with ethyl acetate. The ethyl acetate phaseswere combined and dried over sodium sulfate, then carefully concentratedto dryness. Crude product was purified on a well-equilibrated andneutralized silica column (1.5×75 cm), elutingtriethylamine/cycclohexane/ethylacetate (1:50:150). The resultingproduct was concentrated to dryness, and lyophilized frompyridine/benzene (1:99) to give product nucleoside amidite as a fluffywhite solid (2.6 g, 72% recovery) which had the following chacteristics:

UV (methanol): λ max 302 nm (ε 18,400), 288 nm, 236 nm (ε 25,700)

TLC (plates prewashed TEA/EtOAc 1:49): Rf=0.45, 0.55 (diasteriomers)(EtOAc/cyclohexane 2:1).

Elemental analysis (C, H, N, P, F): +0.6%. P-NMR: S 149.1, 149.5(diasteriomers).

This modified nucleoside amidite most closely resembles thymidine instructure and in hybridization characteristics, and can be substitutedin place of one or more thymidines for oligonucleotide synthesis.

EXAMPLE XXX Preparation of5'-DMT-5-[N-trifluoroacetylaminoheptyl)1-acrylamido]-2'-deoxyuridineBeta-cyanoethyl N,N-diisopropylphosphoramidite

The procedure of Example XXIX was followed usingN,N-diisopropyl-(2-cyanoethyl)phosphoramidic chloride as thephosphitylating agent. The resulting product had the followingcharacteristics:

UV: identical to that of product of Example XXIX

TLC: (plates prewashed TEA/EtOAc 1:49) Rf=0.35 0.48 (diasteriomers)(EtOAc/cyclohexane 2:1))

Elemental Analysis (C, H, N, P, F): ±0.3%

EXAMPLE XXXI Synthesis of Linker Arm Oligonucleotides by PhosphoramiditeMethodology

Phosphoramidite synthesis was accomplished using an Applied Biosystems,Inc. (ABI) model 380A programmable DNA synthesizer in accordance withthe manufacturer's instructions. As is well known, DNA synthesizersselectively mix different synthesis reagents according to apredetermined time and volumetric program. To achieve this, the severalreagents were stored in containers, each having a valved exit to amixing chamber. The outlet valves were selectively and automaticallyopened to admit the reagent to the mixing chamber in accordance with acomputer controlled sequence. The program utilized was basedsubstantially on the phosphoamidite synthesis protocol of Caruthers, et.al. (1981). J. Am. Chem. Soc. 103:3185. Prior to synthesis, 20 mg foreach condensation of modified nucleoside amidite as prepared in ExamplesXXIX or XXX was weighed into the amidite bottle, lyophilized frompyridine/benzene overnight, and dissolved in 150 μl dry acetonitrile per20 mg modified nucleoside amidite. The modified nucleoside amiditebottle was connected to the synthesizer and the desired sequences,chosen to be complementary to the nucleic acid sequence chosen forhybridization, programmed into the machine. The desired programsubstitutes the modified nucleoside amidite in place of one or morethymidines. Coupling efficiencies of the modified nucloetide wereindistinguishable from standard amidites, and averaged greater than 98%by measurement of DMT releases. To allow purification of the productoligonucleotide by HPLC, the chosen program left DMT attached to theproduct.

After synthesis was completed, standard cleavage methods (1 hourthiophenol for methyl amidites, ammonium hydroxide treatment) were used.Deprotection in ammonium hydroxide was 2 hours at ambient temperature,then 50° C. for 15 hours. The crude oligonucleotide solution wasconcentrated carefully to a small volume (<500 μl) in the presence oftributylamine. The DMT crude was analyzed by analytical reverse phasehigh pressure liquid chromatography (RPHPLC) and purified by preparatoryRPHPLC. Those fractions containing the DMT-oligomer were combined,concentrated to dryness, and treated with 80% acetic acid for 40 minutesat ambient temperature to remove DMT. The product was desalted on a 1×30cm column of Sephadex RG-25 (fine), (Pharmacia Fine Chemicals,Piscataway, N.J.) and ethanol precipitated from 300 mM sodium acetate,pH 7. Analysis by PAGE both by kinasing and autoradiography, and byStains-all (Aldrich Chemical Co., Milwaukee, Wis.) developed gelsindicating a single homogeneous band.

EXAMPLE XXXII Preparation of Fluorescent Linker Arm Oligonucleotides

To a solution of 300 μg linker arm oligonucleotide (8.1 OD260, 50 nmol,prepared by the method of Example XXXI) in 250 μl 500 mM sodiumbicarbonate, pH 9.4, was added 8.6 mg FITC solid. The solution wasagitated at room temperature for 6 hours. The product oligonucleotidewas separated from unreacted FITC using 1×30 cm Sephadex® G-25 columneluting 20 mM ammonium acetate, pH 6. Fractions in the firstUV-absorbing peak were combined. Analysis by analytical 20% PAGEindicated reaction was complete, with fluroescent oligomerelectrophoresing slower by the equivalent of 1 nucleotide unit. TheFITC-oligomer was purified by preparatory RPHPLC detectingsimultaneously at 260 and 495 nm. The product was concentrated andethanol precipitated to recover 4.8 OD260 (55%) of fluoresceinatedoligonucleotide. Products were found to be homogeneous by PAGE andRPHPLC, to kinase normally, and to have UV-vis absorbance ratiospredicted for specific oligomer-fluorophore conjugates. Thesefluorescent linker arm oligonucleotides are used directly forhybridization to nucleic acids to determine the presence ofcomplementary sequence.

EXAMPLE XXXIII Preparation Of Linker Arm Oligonucleotide-alkalinePhosphatase Conjugates

Twenty-five microliters of linker arm oligonucleotide (14 nMol) at aconcentration of 4 mg/ml in 0.1M sodium bicarbonate and 2 mM EDTA wascombined with 50 μl solution of disuccinimidyl suberate (DSS) (1.4 μmol)at a concentration of 10 mg/ml in dimethyl sulfoxide. The reaction wasallowed to proceed for 5 minutes at room temperature in the dark, thenimmediately applied to a Sephadex® G-25 column, 1 cm×40 cm, and elutedat 4° C. with water. The eluted fractions (0.5 ml) were monitored byabsorbance at 260 nm. The first peak fractions, containing activatedlinker arm oligonucleotide, were pooled and frozen for lyophilization asquickly as possible to minimize hydrolysis of reactive succinimidylgroups. Unreacted DSS and products were well resolved from the modifiedoligomer fractions. The lyophilized and modified linker arm oligomer wasrehydrated with a two-fold stoichiometric excess of alkaline phosphatase(4 mg) in 200 μl of 0.1M sodium bicarbonate, 3M NaCl, 0.05% sodiumazide, pH 8.25. The conjugation reaction mixture was maintained at roomtemperature for 16 hours. The products of the conjugation reaction wereseparated by gel filtration chromatography using a 1×100 cm column ofP-100 (Bio Rad, Richmond, Calif.) eluting 50 mM Tris, pH 8.5, at 4° C.The protein containing fractions were pooled and dialyzed against 50 mMtris, pH 8.5, in the cold. Pure oligomer-alkaline phosphatase conjugatewas obtained by chromatography on a 1×6 cm column of DEAE celluloseeluting 0.1 mM Tris in 0.1M NaCl. The peak fractions were pooled andconcentrated to approximately 1 mg/ml protein by vacuum dialysis against50 mM tris, pH 8.5, and stored at 4° C. in the presence of 0.05% sodiumazide. Alkaline phosphatase retained full enzymatic activity, 400-500U/mg protein, as determined by spectrophotometric assay, throughout theconjugation and purification processes. The overall yield with respectto oligomer was approximately 30-50%.

Although the invention has been described with reference to specificexamples, it should be understood that various modifications can be madewithout departing from the spirit of the invention. Accordingly theinvention is limited only by the following claims.

I claim:
 1. A process for chemically synthesizing a modifiedsingle-stranded oligonucleotide comprising:adding at a preselectedposition in said oligonucleotide a modified nucleotide compound havingthe structure: ##STR22## wherein B is a pyrimidine or purine base and Ris a linker arm including a blocked functional group, and providedthatwhen R₄ is a blocking group thenR₅ is a reactivephosphorous-containing group or is a hydrogen atom if the 5'-hydroxylgroup of the 5'-terminal nucleotide of a growing oligonucleotidecontains a reactive phosphorous-containing group; and when R₅ is ablocking group thenR₄ is a reactive phosphorous-containing group or is ahydrogen atom if the 3'-hydroxyl group of the 3'-terminal nucleotide ofa growing oligonucleotide contains a reactive phosphorous-containinggroup; and R₈ is a hydrogen atom or a blocked hydroxyl group, whereinsaid addition is carried out under conditions to effect coupling of thereactive phosphorous-containing groups of said modified nucleotide to areactive 3'- or 5'-hydroxyl of the oligoncleotide while not impairingthe reactivity of the protected reactive functionality on the linker armR; and, if required, subsequently removing the blocking group from R₄ orR5 and coupling one or more additional modified or unmodified nucleotideunits until the synthesis of said oligonucleotide is complete.
 2. Theprocess of claim 1 wherein said blocked functional group comprises afunctional group and a blocking group, and wherein said process furthercomprises removing said blocking group and reacting said functionalgroup with a reporter group.
 3. The process of claim 2 wherein saidreporter group is selected from the group consisting of a detectablelabel and a member of a specific binding ligand pair.
 4. The process ofclaim 3 wherein said reporter group is a detectable label selected fromthe group consisting of colorimetric, spectrophotometric, fluorometricand radioactive labels.
 5. The process of claim 3 wherein said reportergroup is one member of a specific binding ligand pair selected from thegroup of ligand pairs consisting of antigen-antibody,carbohydrate-lectin, protein-receptor, biotin-avidin, DNA-DNA andRNA-RNA.
 6. The process of claim 5 wherein said reporter group is astrand of DNA.
 7. The process of claim 1 wherein said preselectedposition is at a terminal position of the oligonucleotide.
 8. Theprocess of claim 1 wherein said preselected position is at an internalposition of the oligonucleotide.
 9. The process of claim 1 wherein R₅ isa reactive phosphorous-containing group which is coupled to a reactive5'-hydroxyl group.
 10. The process of claim 1 wherein the linker arm Ris attached at a sterically tolerant site of the base.
 11. The processof claim 10 wherein B is a pyrimidine base and R is attached at the C₅position of the base.
 12. The process of claim 10 wherein B is a purinebase and R is attached at the C₈ position of the base.
 13. The processof claim 12 wherein R is attached through a polyvalent heteroatomselected from the group consisting of nitrogen, oxygen and sulfur. 14.The process of claim 1 wherein said blocked functional group comprises afunctional group and a blocking group, and wherein said process furthercomprises removing said blocking group and reacting said functionalgroup with a solid support.
 15. A process for chemically synthesizing amodified single-stranded oligonucleotide comprising:adding at apreselected position in said oligonucleotide a modified nucleotidecompound having the structure: ##STR23## wherein B is a pyrimidine orpurine base and R is a linker arm covalently attached to a detectablelabel or solid surface that does not adversely affect the couplingreaction, and provided thatwhen R₄ is a blocking group thenR₅ is areactive phosphorous-containing group or is a hydrogen atom if the5'-hydroxy group of the 5'-terminal nucleotide of a growingoligonucleotide contains a reactive phosphorous-containing group; andwhen R₅ is a blocking group thenR₄ is a reactive phosphorous-containinggroup or is a hydrogen atom if the 3'-hydroxyl group of the 3'-terminalnucleotide of a growing oligonucleotide contains a reactivephosphorous-containing group; and R₈ is a hydrogen atom or a blockedhydroxyl group, wherein said addition is carried out under conditions toeffect coupling of the reactive phosphorous-containing group of saidmodified nucleotide to a reactive 3'- or 5'-hydroxyl of theoligoncleotide; and, if required, subsequently removing the blockinggroup from R₄ or R₅ and coupling one or more additional modified orunmodified nucleotide units until the synthesis of said oligonucleotideis complete.
 16. The process of claim 15 wherein R5 is a reactivephosphorous-containing group which is coupled to a reactive 5'-hydroxylgroup.
 17. The process of claim 15 wherein the linker arm R is attachedat a sterically tolerant site of the base.
 18. The process of claim 17wherein B is a pyrimidine base and R is attached at the C₅ position ofthe base.
 19. The process of claim 17 wherein B is a purine base and Ris attached at the C₈ position of the base.
 20. The process of claim 19wherein R is attached through a polyvalent heteroatom selected from thegroup consisting of nitrogen, oxygen and sulfur.